Process for continuously preparing bacterial cellulose

ABSTRACT

An object of the present invention is to obtain the high production rate and yield of BC. The present invention relates to a process for the production of cellulosic material at a production rate of 0.4 g/L/hr or more, which comprises culturing cellulose-producing bacteria while maintaining the concentration of the residual sugars in a culture broth at 20 g/L or less, and to a process for the production of cellulosic material at a production rate of 0.4 g/L/hr or more, which comprises culturing cellulose-producing bacteria in a culture medium containing a factor which improves an apparent affinity of substrate for sugars.

TECHNICAL FIELD

This invention relates to a process for the production of cellulosicmaterial (bacterial cellulose: "BC") at a high rate, which comprisesusing microorganisms capable of producing BC (cellulose-producingbacteria) while maintaining the concentration of the residual sugars ina culture broth at a specific level or less.

BACKGROUND ART

Since BC is edible, it is utilized in the food industry. BC's highdispersibility in water further provides it with a lot of industrialapplications, such as to maintain viscosity of food, cosmetics orcoating agents, to strengthen food materials, to maintain moisture, toimprove stability of food, and to be used as low-calorie additives andas an emulsion stabilizer.

BC is characterized by a sectional width of its fibrils, which issmaller by two orders of magnitude than that of other kinds of cellulosesuch as those derived from wood pulp.

Owing to such structural and physical features of microfibrils, ahomogenized BC has plenty of industrial applications as a strengtheningagent for polymers, especially hydrophilic polymers. Products preparedby solidification of the homogenized BC in the form of a lump or papershow a high elastic modulus in tension owing to the above feature, andare therefore expected to have excellent mechanical properties for usein various kinds of industrial materials.

Methods for the production of BC are described in, for example, JapanesePatent Laid-Open Application Sho 62(1987)-265990, Japanese PatentLaid-Open Application Sho 63(1988)-202394 and Japanese PatentPublication Hei 6(1994)-43443.

As a nutrient medium suitable for the cultivation of thecellulose-producing bacteria, Schramm/Hestrin medium is known, whichcontains carbon source, peptone, yeast extract, sodium phosphate andcitric acid (Schramm et al., J. General Biology, 11,pp.123-129, 1954).Further, it has been found that the productivity of the BC is increasedby the addition of an accelerator for the cellulose production such asinositol, phytic acid and pyrroloquinoline quinone (PQQ) (JapanesePatent Publication Hei 5(1993)-1718; Mitsuo TAKAI, Japan TAPPI Journal,Vol.42, No.3, pp.237-244), carboxylic acid or their salts (JapanesePatent Laid-Open Application Hei 5(1993)-191467; Japanese PatentLaid-Open Application Hei 7(1995)-39386), invertase (Japanese PatentApplication Hei 5(1993)-331491; Japanese Patent Laid-Open ApplicationHei 7(1995)-184677) and methionine (Japanese Patent Application Hei5(1993)-335764; Japanese Patent Laid-Open Application Hei7(1995)-184675) into such a nutrient medium.

Furthermore, there have been proposed a method for cultivating thecellulose-producing bacteria under a specific range of oxygen-transfercoefficient (K_(L) a) (Japanese Patent Application Hei 7(1995)-31787), amethod for cultivating the cellulose-producing bacteria whilemaintaining the concentration of carbon sources in a culture broth at aspecific level or more (Japanese Patent Application Hei 7(1995)-267407)and a method for cultivating the cellulose-producing bacteria whilemaintaining the internal pressure within a fermentation tank at aspecific level or more at a certain stage during the cultivation(Japanese Patent Application Hei 7(1995)-76408).

The bacteria may be generally cultured in any known culture conditionssuch as static culture, shaken culture, and aerated and agitatedculture, and in any known culture operation methods such as batchfermentation, fed batch fermentation, repeated batch fermentation andcontinuous fermentation.

Means for agitation include impellers (agitating blades), air-liftfermenters, pump-driven recirculation of the fermenter broth and anycombination of these means.

The impellers include gate-shape impellers, turbine impellers, doublehelical ribbon impellers and screw impellers.

An economical and high-yielding method for the production of bacterialcellulose is described in Japanese Patent Laid-Open Application Hei8(1996)-33495, wherein the concentration of the bacterial cellulose in aculture medium is kept at a specific level or less or the oxygenconsumption rate is kept at a specific level or more by a continuousremoval of the culture medium from its culture system and a continuoussupply of a fresh culture medium having almost the same volume as theremoved culture broth.

The above method has overcome the disadvantages that accumulation of BCin the culture broth during the culture of the cellulose-producingbacteria will increase viscosity of the culture broth and make itdifficult to supply a necessary amount of oxygen into the culture broth.As a result, a high production rate has been attained. However, since adilution rate (i.e., a rate of supply of the medium) is relatively high,sugars will remain unused in the withdrawn culture broth, leaving aproblem to be solved in the yield of BC production.

The present inventors have studied to solve the above problem so as tomake the present invention.

DISCLOSURE OF INVENTION

The present invention relates to a process for the production ofcellulosic material at a production rate of 0.4 g/L/hr or more, whichcomprises culturing cellulose-producing bacteria while maintaining theconcentration of the residual sugars in a culture broth at 20 g/L orless, preferably at 8 g/L or less.

In the present method, the culture broth containing BC is continuouslydrawn from its culture system while maintaining the concentration of theresidual sugars in the culture broth by controlling the dilution rate.As a result, BC yield may be increased in the continuous cultivation ofcellulose-producing bacteria.

The term "continuous" in the present specification also contains themeaning of "intermittent" or "off and on." Thus, this concept includesnot only an embodiemnt wherein the supply of the culture medium andwithdrawing of the culture broth in its culture system is successivelycarried out at a constant flow rate by means of a peristatic pump andthe like, but also an embodiment wherein the supply of the culturemedium and withdrawing of the culture broth is carried out at a certaininterval.

In any case, the concentration of the residual sugars in the culturebroth may be maintained at a low level by determining the amount ofsupply and withdrawing depending on a monitored value of theconcentration of the residual sugars in the withdrawn culture broth.

One example of maintaining the concentration of the residual sugars inthe culture broth at 20 g/L or less may be to control the dilution rateat 0.12/hr or less in the case of using BPR 3001A strain as thecellulose-producing bacteria. The "dilution rate" is defined as followsin the present specification:

    D=F/V

wherein

D: Dilution rate (/hr),

F: Supply rate of the culture medium and

V: Amount of the culture broth within a fermentation tank.

Further, the present invention relates to a method for the production ofcellulosic material, which comprises culturing cellulose-producingbacteria in a culture medium containing a factor which improves anapparent affinity of bacteria for sugars substrate such as fructose ordecreases an affinity constant of substrate (Ks).

The factor includes lactic acid, ethanol, acetaldehyde, acetic acid,pyruvic acid and glycerol as well as the combination thereof.

The concentration of the factors in the medium may be optionallydetermined by those skilled in the art considering culturing conditionssuch as the kinds of bacteria to be cultured and culture medium and thelike, being usually in the range of 1-30 g/L.

The "affinity constant of substrate (Ks)" means the affinity between thebacteria and a substrate, and is equal to the value of the concentrationof the substrate when "half the maximum value of a specific growth rate(μ_(max))" is shown. Thus, the higher the affinity of substrate for acertain substrate is, the smaller the value of affinity constant ofsubstrate (Ks) becomes. According to Monod's equation, the followingrelation is found among the affinity constant of substrate (Ks), themaximum value of a specific growth rate (μ_(max)), a specific growthrate (μ) and the concentration of the remaining substrates (S):

    μ=μ.sub.max S/(Ks+S)

Since the dilution rate (D) may be considered to be equal to thespecific growth rate (μ) at a steady state in the continuouscultivation, the value of the affinity constant of substrate (Ks) of aparticular bacterium will be empirically calculated by means ofLineweaver-Burk plot for a given substrate, based on the relationshipbetween the concentration of the substrate in the culture broth anddilution rate at the steady state.

In addition to the above culture conditions and culture operationmethods, it is also possible to use for the present invention the methodfor the production of BC described in the Japanese Patent ApplicationHei 6(1994)-192287 (Japanese Patent Laid-Open Application Hei8(1996)-33494), wherein a culture broth containing bacteria iscirculated between a cultivating apparatus and a separator such as afloatation equipment and an edge filter to separate the resulting BCfrom the bacteria and culture broth in said separator.

The cellulose-producing bacteria used in the present invention includeAcetobacter strains such as Acetobacter xylinum subsp. sucrofermentanssuch as BPR 2001 strain, Acetobacter xylinum ATCC23768, Acetobacterxylinum ATCC23769, Acetobacter pasteurianus ATCC10245, Acetobacterxylinum ATCC14851, Acetobacter xylinum ATCC11142, Acetobacter xylinumATCC10821; Agrobacterium; Rhizobium; Sarcina; Pseudomonus,Achromobacter; Alcaligenes; Aerobacter; Azotobacter; and Zooglea; andstrains derived from those strains by using known mutagens such as NTG(nitrosoguanidine).

The BPR 2001 was deposited at the National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology (1-3,Higashi 1-chome, Tsukuba-shi, Ibaraki-ken 305 Japan) on Feb. 24, 1993under accession number FERM P-13466, and then transferred on Feb. 7,1994 to the deposit under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure and Regulation under accession number FERMBP-4545.

The chemical treatment using mutagens such as NTG is described in, forexample, Bio Factors, Vol. 1, pp.297-302 (1988) and J. Gen. Microbiol,Vol. 135, pp.2917-2929 (1989). Accordingly, those skilled in the art mayobtain the present mutants in accordance with these known methods. Thepresent mutants may be also obtained by other treatments such asapplication of radioactive rays.

Among the thus obtained mutants, it is preferred to usecellulose-producing bacteria capable of producing a bacterial cellulosehaving a weight-average degree of polymerization (in terms ofpolystyrene) of 1.6×10⁴ or above, preferably of 1.7×10⁴ or above in anaerobic agitated culture, or cellulose-producing bacteria capable ofproducing a bacterial cellulose having a weight-average degree ofpolymerization (in terms of polystyrene) of 2.0×10⁴ or above in a staticculture.

One example of the present cellulose-producing bacteria, BPR3001A, hasbeen deposited at the National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology (1-3,Higashi 1-chome, Tsukuba-shi, Ibaraki-ken 305 Japan) on Jun. 12, 1995under accession number FERM P-14982, and then transferred on Feb. 23,1996 to the deposit under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure and Regulation under accession number FERMBP-5421.

It is well known that strength and elasticity of polymeric materials areimproved as their degree of polymerization increases. As that is alsothe case with a bacterial cellulose, membranes prepared from bacterialcellulose having a high degree of polymerization will show higherstrength and elasticity than membranes prepared from bacterial cellulosehaving a relatively low degree of polymerization. Accordingly, by usingthe bacterial cellulose with a high degree of polymerization, themembrane with a high strength and elasticity may be obtained in thepresent invention.

The weight-average degree of polymerization of a variety kinds ofcellulose such as BC of this invention may be determined by the methodusing a GPC system (Tosoh HLC-8020) equipped with an RI detector asfollows:

A cellulose sample is nitrated with a fuming nitric acid-phosphorouspentaoxide solution according to the method of W. J. Alexander, R. L.Mitchell, Analytical Chemistry 21, 12, 1497-1500 (1949).

Nitrated cotton linter is used as a control.

Nitrated cellulose is then dissolved in THF (Wako Pure ChemicalIndustries Ltd., the first grade) to a final concentration of 0.05%, andfiltered through a 1.0 μm pore-size filter. THF is also used for anelution solvent.

The flow rate, pressure, and sample-injection volume are adjusted to be0.5 ml/min., 10˜13 kgf/cm² and 100 μl, respectively.

The column system consists of two TSKgel GMH-HR (S) columns (7.5 ID×300mm) and a guard column (Tosoh Co., Ltd.). The analysis is carried out ata temperature of 35° C.

A relative molecular weight in terms of polystyrene is calculated byusing polystyrene standards (Tosoh).

The polystyrene standards having a molecular weight in the range of2.0×10⁷ to 2630 are used and a standard curve is prepared based on thefollowing three-dimension approximate equation:

    log M=At.sup.3 +Bt.sup.2 +Ct+D

wherein "t" is an elution time and "M" is a molecular weight.

The weight-average molecular weight and number-average molecular weightare calculated by a program (ver. 3, 10) equipped in a data processor(SC-8020).

The weight-average degree of polymerization and number-average degree ofpolymerization of the original cellulose samples are finally calculatedbased on the above data, taking substitution degrees after the nitrationinto consideration.

Carbon sources in the culture media useful in the present inventioninclude sucrose, glucose, fructose, mannitol, sorbitol, galactose,maltose, erythritol, glycerol, ethyleneglycol, ethanol and theirmixtures. In addition, sucrose may be combined with starch hydrolysatecontaining these carbon sources, citrus molasses, beet molasses,squeezed juice from beet or sugar cane, juice from citrus and the like.

Nitrogen sources useful in the present invention include organic orinorganic substances such as ammonium salts including ammonium sulfate,ammonium chloride, ammonium phosphate; nitrates; and urea.Nitrogen-containing natural nutrients may be also used includingBacto-Peptone, Bacto-soytone, Yeast-Extract, and Bean-Condensate.

A trace amount of organic nutrients may be further added including aminoacids, vitamins, fatty acids, nucleic acids, 2,7,9-tricarboxy-1Hpyrrolo[2,3,5]-quinoline-4,5-dione, sulfite pulp waste liquor, ligninsulfonic acid and the like.

When the mutants with nutritional requirement for amino acids are used,such required nutrients should be supplemented in the culture media.Inorganic nutrients include phosphate salts, magnesium salts, calciumsalts, iron salts, manganese salts, cobalt salts, molybdate salts,hematite salts, chelate metal salts and the like.

It is also possible to optionally supply the abovementioned acceleratorsfor the cellulose production.

For example, when the Acetobacter is used as the cellulose-producingbacteria, a pH range for the culture is controlled between 3 and 7,preferably around 5. A culture temperature is kept in a range between 10and 40° C., preferably between 25 and 35° C. Oxygen supply into acultivating apparatus may contain from 1 to 100% oxygen, desirably 21 to80%. Those skilled in the art may optionally determine the contents ofthese components in the culture media and the inoculation of thebacteria into the media, depending on the culture method to be used.

The BC produced in the present method may be recovered together with thebacterial cells, and then impurities other than the BC, including thebacterial cells per se, may be removed from the recovered BC.

The impurities may be almost completely removed from the BC by washing,dehydration under pressure, dilute acid washing, alkali washing,bleaching with hypochlorite soda or hydrogen peroxide, lysing with lyticenzymes such as lysozyme, treatment with surfactants such as laurylsulfate soda or deoxycholate soda, washing under heat at a temperaturerange between a room temperature and 200° C., and any combination ofthese treatments.

The BC thus obtained according to the present invention includescellulose, those comprising heteropolysugars having cellulosic mainchains, and those comprising β-1,3- or β-1,2-glucan. Saidheteropolysugars contain as components hexoses, pentoses and organicacids such as mannose, fructose, galactose, xylose, arabinose, rhamnoseand glucuronic acid, as well as glucose.

These polysugars may be present alone or as a mixture combined eachother via hydrogen bonds.

BRIEF DESCRIPTION OF DRAWING

FIG. 1 shows the relationship among the dilution rate, the productionrate of BC and the concentration of the residual sugars in the drawnbroth.

FIG. 2 shows the relationship among the dilution rate, the yield againstthe consumed sugars and the yield against the supplied sugars.

FIG. 3 plots reciprocal numbers (1/S) and (1/D) of the concentration ofthe residual substrates (sugars) (S) and the concentration of thedilution rate (D), repectively.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be further illustrated according to thefollowing examples.

EXAMPLE 1

BPR 3001A strain was continuously cultured under the followingconditions.

Cultivating Condition

A conical flask with slanted baffles (500 ml volume) containing 112.5 mlof CSL-Fru medium shown in Table 1 was inoculated with a Roux flaskculture mixture (12.5 ml), and incubated for three days at 28° C. withshaking at 180 rpm. The resulting culture mixture was asepticallyhomogenized with a homogenizer for 1 min at 10,000 rpm. 12.5 ml of thethus homogenized culture mixture was divided and inoculated into sixconical flasks with slanted baffles (500 ml volume each) containing112.5 ml each of CSL-Fru medium shown in Table 1 and cultured for 24hours at 28° C. with shaking at 180 rpm.

The resulting culture mixture (160 ml) was then inoculated into each ofthree jars (3L volume each) containing 1440 ml each of CSL-Fru mediumshown in Table 1 and subjected to a batch fermentation. The startingconcentrations of sugars and CSL were 40 g/L and 4 v/v %, respectively.A pH range for the culture is controlled at 5+0.1 and a culturetemperature is kept at 30° C. An amount of aeration was maintained at800 ml/min, and a rate of agitation was kept in the range between 200and 1200 rpm so that an amount of dissolved oxygen could be satisfied.

The following three kinds of culture media were used:

(1) CSL-Fru medium shown in FIG. 1 with an exception of theconcentration of the sugars of 30 g/L;

(2) The above medium in (1) further supplemented with 7.5 g/L of lacticacid; and

(3) The above medium in (1) further supplemented with 10 g/L of ethanol.

During the BC production, each of the above three media was supplied for36 hours at the dilution rate of 0.034 (/hr), 0.069 (/hr) 0.102 (/hr),respectively, after 20 hours had passed since the initiation of culture.The amounts of the supplied culture medium and withdrawn culture brothwere weighed and controlled so as to be the same with each other.

The resulting relationship among the dilution rate, the production rateof BC and the concentration of the residual sugars in the withdrawnbroth, and the relationship among the dilution rate, the yield againstthe consumed sugars and the yield against the supplied sugars are shownin FIG. 1 and FIG. 2, respectively.

                  TABLE 1                                                         ______________________________________                                        CSL-Fru medium                                                                ______________________________________                                        Fructose            4.0 (%)                                                                        KH.sub.2 PO.sub.4 0.1                                      MgSO.sub.4.7H.sub.2 O 0.025                                                   (NH.sub.4).sub.2 SO.sub.4 0.33                                                Vitamin Mixture (see below) 1.0                                               Salt Mixture (see below) 1.0                                                  CSL (Corn Steep Liquor) 4.0                                                   pH 5.0                                                                      ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Vitamin Mixture                                                                       compound       mg/L                                                   ______________________________________                                        Inositol           200                                                          Niacin 40                                                                     Pyridoxine HCl 40                                                             Thiamine HCl 40                                                               Ca Pantothenate 20                                                            Riboflavin 20                                                                 p-Aminobenzonic Acid 20                                                       Folic Acid 0.2                                                                Biotin 0.2                                                                  ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Salt Mixture                                                                  ______________________________________                                        FeSO.sub.4.7H.sub.2 O                                                                        360 (mg/L)                                                       CaCl.sub.2.2H.sub.2 O 1470                                                    Na.sub.2 MoO.sub.2.2H.sub.2 O 242                                             ZnSO.sub.4.7H.sub.2 O 173                                                     MnSO.sub.4.5H.sub.2 O 139                                                     CuSO.sub.4.5H.sub.2 O 5                                                     ______________________________________                                    

The concentration of the residual sugars in the withdrawn culture brothwas determined by subjecting a sample of the withdrawn culture brothappropriately diluted and filtered through cellulose acetate membrane of0.45 μm to high performance liquid chromatography (Nihon Bunko Co., Ltd.HPLC separation column: Shodex Asahipak NH2P-50 4E).

The accumulated amount of BC (g/L) was calculated for obtaining theproduction rate of BC in the continuous cultivation as follows. Thesolid contents in the withdrawn culture broth were collected, washedwith water to remove the medium components, and treated with 1 N NaOHaqueous solution at 80° C. for 20 minutes to remove the bacterial cells.The resulting cellulose was washed until the washing water becameapproximately neutral, and dried under vacuum at 80° C. for 12 hours toweigh the dry cellulose.

The yield against the consumed sugars (%) and the yield against thesupplied sugars (%) were calculated as follows.

Calculation of Yield Aagainst the Consumed Sugars (%)

In the continuous cultivation system wherein a culture medium having theconcentration of sugars (Fru_(in) (g/L)) is supplied at the rate ofsupply (F(L/hr)), there is the culture broth of V (L) having theconcentration of BC (BC₀ (g/L)) and the concentration of sugars (Fru₀(g/L)) at the time of t₀. Then at the time of t₁, the concentration ofBC and the concentration of sugars have changed to (BC₁ (g/L)) and (Fru₁(g/L)), respectively. During the time course from t₀ to t₁, v (L) of theculture broth was withdrawn, wherein the concentration of BC is BC_(out)(g/L) and the concentration of the residual sugars is Fru_(out) (g/L).

The yield against the consumed sugars Yc (%) is represented as follows:

    Yc=(P.sub.1 -P.sub.0 +P.sub.out)/(S.sub.0 +S.sub.in -S.sub.1 -S.sub.out)*100

wherein

Yc: Yield against the consumed sugars (%)

P₀ : Total amount of BC in the fermenter at t₀ (g) (P₀ =BC₀ *V)

P₁ : Total amount of BC in the fermenter at t₁ (g) (P₁ =BC₁ *V)

P_(out) : Total amount of BC in the culture broth withdrawn during thetime from t₀ to t₁ (g) (P_(out) =BC_(out) *V)

S₀ : Total amount of sugars in the fermenter at t₀ (g) (S₀ =Fru₀ *V)

S₁ : Total amount of sugars in the fermenter at t₁ (g) (S₁ =Fru₁ *V)

S_(in) : Total amount of sugars supplied from t₀ to t₁ (g) (S_(in)=Fru_(in) *v)

S_(out) : Total amount of sugars in the culture broth withdrawn duringthe time from t₀ to t₁ (g) (S_(out) =Fru_(out) *v)

t: Course of time (hr) (t=t₁ -t₀)

v: Amount of the culture broth withdrawn during the time from to t₀ t₁(v=F*t)

Calculation of Yield Against the Supplied Sugars (%)

The yield against the supplied sugars Yt (%) is represented as follows:

    Yt=(P.sub.1 -P.sub.0 +P.sub.out)/S.sub.in * 100

wherein

Yt: Yield against the supplied sugars (%)

P₀ : Total amount of BC in the fermenter at t₀ (g) (P₀ =BC₀ *V)

P₀ : Total amount of BC in the fermenter at t₁ (g) (P₁ =BC₁ *V)

P_(out) : Total amount of BC in the culture broth withdrawn during thetime from t₀ to t₁ (g) (P_(out) =BC_(out) *v)

S_(in) : Total amount of sugars supplied from t₀ to t₁ (g) (S_(in)=Fru_(in) *v)

t: Course of time (hr) (t=t₁ -t₀)

v: Amount of the culture broth withdrawn during the time from t₀ to t₁(v=F*t)

REFERENCE EXAMPLE

The relationship between the dilution rate (D) and the concentration ofthe residual sugars in the withdrawn culture broth (S) was studied atthe steady state of the continuous cultivation using the three differentsupplying culture media.

According to the following modified Monod's equation, the reciprocalnumbers (1/S) and (1/D) of the concentration of the residual substrates(sugars) (S) and the concentration of the dilution rate (D),repectively, are plotted in FIG. 3.

    1/μ=1/D=(Ks/μ.sub.max)*(1/S)+(1/μ.sub.max)

Segment in the X-axis obtained by the method of least squares in theabove analysis was equal to (-1/Ks). The results are shown in Table 4.

Table 4 demonstrates that the addition of ethanol or lactic acid reducesthe value of the affinity constant of substrate (Ks), meaning theimprovement of an apparent affinity for sugars.

                  TABLE 4                                                         ______________________________________                                        Apparent value of the constant of affinity (Ks) of                              BPR 3001A strain for fructose in the continuous cultivation                     Medium         Constant of affinity of substrate (Ks)                     ______________________________________                                        CSL-Fru (control)                                                                            9 (g/L)                                                          CSL-Fru plus Ethanol 5.6                                                      CSL-Fru plus Lactic acid 3.3                                                ______________________________________                                    

INDUSTRIAL APPLICABILITY

As seen form the results shown in FIG. 1, by the addition of the factorsuch as ethanol or lactic acid which improves an apparent affinity ofsubstrate of the bacteria for sugars in the culture broth according tothe present invention, the advantages may be effected that theproduction rate of BC is increased while the concentration of theresidual sugars in the drawn culture broth is decreased at the samedilution rate in the continuous cultivation of the cellulose-producingbacteria, when compared with the case using as the control CSL-Frumedium containing no such factors.

FIG. 2 shows that the above addition may further improve both the yieldagainst the consumed sugars and yield against the supplied sugars.

What is claimed is:
 1. A process for the production of cellulosicmaterial at a production rate of 0.4 g/L/hr or more in a culture system,which comprises:continuously culturing cellulose-producing bacteriawhile continuously removing culture medium from the system containingthe culture medium and continuously supplying fresh culture mediumthereto such that the concentration of sugars in the withdrawn culturemedium in said system is maintained at no more than 20 g/L.
 2. Theprocess of claim 1, wherein the concentration of sugars in the culturebroth is 8 g/L or less.
 3. The process of claim 1, wherein saidcellulose-producing bacteria are selected from the group consisting ofgenera of Acetobacter, Agrobacterium, Rhizobium, Sarcina, Pseudomonous,Achromobacter, Alcaligenes, Aerobacter, Azotobacter, Zooglea and strainsderived from these bacterial strains employing known mutagens.
 4. Theprocess of claim 1, wherein said cellulosic material has a weightaverage degree of polymerization of 1.6×10⁴ or above.
 5. A process forthe production of cellulosic material at a production rate of 0.4 g/L/hror more in a culture system, which comprises:continuously culturingcellulose-producing bacteria while continuously removing culture mediumfrom the system containing the culture medium and continuously supplyingfresh culture medium thereto such that the culture medium contains afactor which increases the affinity of said bacteria to substratethereby decreasing substrate affinity constant (Ks).
 6. The process forthe production of cellulosic material according to claim 5, wherein thefactor is selected from the group consisting of lactic acid, ethanol,acetaldehyde, acetic acid, pyruvic acid, glycerol and combinationsthereof.
 7. The process according to claim 6, wherein said factor ispresent in the culture medium in a concentration ranging from 7.5 to 30g/L.
 8. The process according to claim 5, wherein said factor is presentin the culture medium in a concentration ranging from 7.5 to 30 g/L. 9.The process according to claim 5, wherein said cellulose-producingbacteria are selected from the group consisting of genera ofAcetobacter, Agrobacterium, Rhizobium, Sarcina, Pseudomonous,Achromobacter, Alcaligenes, Aerobacter, Azotobacter, Zooglea and strainsderived from these bacterial strains employing known mutagens.
 10. Theprocess of claim 5, wherein said cellulosic material has a weightaverage degree of polymerization of 1.6×10⁴ or above.